The present invention relates to inflammatory reactions caused by certain infectious microorganisms. Specifically, it relates to an agent comprising a mucosa-binding molecule linked to a specific antigen derived from a microorganism, which is useful in inducing systemic immunological tolerance to the specific antigen and thus preventing or treating deleterious inflammatory reactions caused by the microorganism.
Introduction of a foreign substance, herein referred to as an antigen (Ag), including a hapten, by injection into a vertebrate organism can result in the induction of an immune response. Typically, an immune response involves the production of specific antibodies (products of B lymphocytes) capable of interacting with the antigen and/or the development of effector T lymphocytes and the production of soluble mediators, termed lymnphokines, at the site of encounter with the antigen. Antibodies and T lymphocytes play essential roles in protecting against a hostile antigen; under certain circumstances, however, they also participate in injurious processes in response to an antigen that lead to destruction of host tissues. For example, the local reaction of antibodies and/or T lymphocytes with an antigen derived from an infectious microorganism at certain anatomical sites can cause extensive tissue damage. This is the case in chronic inflammatory reactions that develop as a result of ineffective elimination of foreign materials, as in certain infections (e.g. tuberculosis, schistosomiasis, and infections caused by Chlamydia, Helicobacter pylori, Pneumocystis carinii, etc.) where immunoproliferative reactions develop at the site(s) of microbial colonization.
To develop vaccines effective against infectious microorganisms that cause destructive immunological reactions, it is desirable, on the one hand, to specifically prevent or reduce the rate of entry of the microorganisms into internal organs (or the uptake of potentially harmful components, such as toxins derived from these microorganisms), and, on the other hand, to specifically suppress (or decrease to an acceptable level) the intensity of deleterious immune processes without affecting the remainder of the immune system.
The most frequent portals of entry of common microbes are the mucosal surfaces covering the digestive tract, the respiratory tract, the urogenital tract, the eye conjunctiva, the inner ear, and the ducts of exocrine glands, which collectively represent the largest (400 m2) organ system in upper vertebrates. Endowed with powerful mechanical and physicochemical cleansing mechanisms, these surfaces are further protected by a specialized immune system that guards them against potential insults from the environment. This system, termed xe2x80x9cmucosa-associated lymphoid tissuexe2x80x9d (MALT), is the largest mammalian lymphoid organ system, and represents a well-known example of a compartmentalized immunological system. Through the compartmentalization of its afferent and efferent limbs, MALT functions essentially independently from the systemic immune apparatus, the latter system comprising peripheral lymphoid organs such as the blood, the bone marrow, the spleen, peripheral lymph nodes, and the thymus. This notion explains why systemic injection of immunogens is relatively ineffective at inducing an immune response in mucosal tissues.
The predominant component of the immune response expressed by MALT is the elaboration of secretory immunoglobulin A (SIgA), the predominant Ig class in human external secretions and one that provides specific immune protection for mucosal tissues. SIgA antibodies provide xe2x80x9cimmune exclusionxe2x80x9d of bacteria and viruses, bacterial toxins, and other potentially harmful molecules, and have also been reported to neutralize certain viruses directly, to mediate antibody-dependent cell-mediated cytotoxicity (in cooperation with macrophages, lymphocytes and eosinophils), and to interfere with the utilization of growth factors for bacterial pathogens in the mucosal environment.
In contrast to the systemic immune apparatus, which is in a sterile compartment and responds vigorously to most invaders, the mucosal immune system guards organs that are replete with foreign matter including microorganisms. It follows that, upon encounter with a given antigen, the mucosal immune system must select appropriate effector mechanisms and regulate the intensity of its response so as to avoid bystander tissue damage and depletion of the immune response capacity.
In addition to inducing local SIgA antibody responses, ingestion or inhalation of antigens (mucosal route) may also result in the development of a state of peripheral immunological tolerance. Tolerance is characterized by a lack of immune responses in non-mucosal tissues when an antigen initially encountered in the digestive tract mucosa or the respiratory mucosa is reintroduced in the organism by a non-mucosal route such as by parenteral injection. Mucosal administration of antigens is in fact a long-recognized method of inducing immunological tolerance (Wells, H., J. Infect. Dis. 9:147, 1911). The phenomenon, often referred to as xe2x80x9coral tolerancexe2x80x9d because it was initially documented by the effect of oral administration of antigen, is characterized by the fact that animals fed or having inhaled an antigen become refractory or have diminished capability to develop a systemic immune response when re-exposed to said antigen introduced by the systemic route, e.g., by injection. In broad terms, application of an antigen onto a mucosal membrane or into a mucosal tissue, be it the intestine, the lung, the mouth, the genital tract, the nose, or the eye, can induce the phenomenon of systemic immunological tolerance. By contrast, introduction of an antigen into a non-mucosal tissue, such as, for example, by a subcutaneous or intravenous route (referred to as systemic immunization) often results in an affirmative systemic immune response.
The phenomenon of xe2x80x9coral tolerancexe2x80x9d is highly specific for the antigen that was introduced by the mucosal route. That is, hypo-responsiveness can only be documented subsequent to injection of the same antigen used to tolerize, but not after injection of a structurally unrelated antigen that had not been encountered previously at mucosal sites.
The specificity of oral tolerance for the initially ingested or inhaled antigen, and the lack of effect on the development of systemic immune responses against other antigens, makes it an increasingly attractive strategy for preventing and treating illnesses associated with or resulting from the development of untoward and/or exaggerated immunological reactions against specific antigens encountered in non-mucosal tissues.
The phenomenon of mucosally induced systemic tolerance may involve all types of immune responses known to be inducible by the systemic introduction of antigen, such as the production of specific antibodies and the development of cell-mediated immune responses to the antigen. Mucosally induced immunological tolerance has therefore been proposed as a strategy to prevent or to reduce the intensity of allergic reactions to chemical drugs (Chase, M. W., Proc. Soc. Exp. Biol. 61:257-259, 1946). It has also been possible in experimental animals and in humans to prevent or decrease the intensity of immune reactions to systemically introduced soluble protein antigens and to particulate antigens such as red cells by the oral administration of red cells (Thomas H. C. et al., Immunology 27:631-639, 1974; Mattingly, J. et al., J. Immunol. 121:1878, 1978; Bierme, S. J. et al., Lancet, 1:605-606, 1979). The phenomenon of mucosally induced systemic tolerance can be utilized to reduce or suppress immune responses not only against foreign antigens but also against self antigens, i.e., components derived from host tissues.
It has also been shown that the enteric administration of schistosome eggs in mice prevented the development or decreased the intensity of hepatic and intestinal granulomatous reactions, which are chronic T cell-mediated inflammatory immune reactions that develop around schistosome eggs during infestation by the parasite Schistosoma (Weinstock, J V et al., J. Immunol. 135:560-563, 1985). Other microorganisms that cause inflammatory (delayed-type hypersensitivity) reactions include Mycobacterium tuberculosis, Mycobacterium avium, Listeria monocytogenes, Brucella abortus, Chlamydia trachomatis, Mycoplasma sp., Porphyromonas (Bacteroides) gingivalis, Helicobacter pylori, Salmonella sp., Shigella sp., Yersinia sp. Cryptosporidium sp., Borrelia sp., Pneumocystis carinii, Candida albicans, Histoplasma capsulatum, Cryptococcus neoformans, Leishmania sp., Plasmodium, Trypanosoma, paramyxoviruses such as respiratory syncytial virus, adenovirus, poliovirus, hepatitis virus, vaccinia and other poxviruses, rhinovirus, herpes simplex virus, variola, and measles virus.
Although the above examples indicate that mucosal administration offoreign antigens offers a convenient way to induce specific immunologic tolerance, its applicability to large scale therapy in human and veterinary medicine remains limited by practical problems. For example, for broad applicability, mucosally-induced immunological tolerance must also be effective in patients in whom the disease process has already established itself and/or in whom potentially tissue-damaging immune cells already exist. This is especially important for patients suffering from (or prone to) a chronic inflammatory reaction to a persistent microorganism. Current protocols for mucosally induced tolerance have had limited success in suppressing the expression of an already established state of systemic immunological sensitization (Hansson, D. G. et al., J. Immunol. 122:2261, 1979).
Most importantly, and by analogy with mucosal vaccines (i.e., preparations used to induce immune responses to infectious pathogens), induction of systemic immunological tolerance by mucosal application of most antigens requires considerable amounts of the tolerogen/antigen, and the tolerance is of relatively short duration, unless the tolerogen/antigen is administered repeatedly over long periods oftime. A likely explanation is that most antigens are extensively degraded before entering a mucosal tissue and/or are absorbed in insufficient quantities. It has thus been widely assumed that only molecules with known mucosa-binding properties can induce local and systemic immune responses when administered by a mucosal route, such as the oral route, without inducing systenmic immunological tolerance (de Aizpurua, H. J. et al., J. Exp. Med. 167:440-451, 1988). Examples of mucosa-binding molecules are listed in Table I below; see also reviews such as Mirelman D., Microbial Lectins and Agglutinins, Properties and Biological Activity, pp. 84-110, Wiley, New York, 1986). A notable example is cholera toxin, one of the most potent mucosal immunogens known so far (Elson, C. O. et al., J. Immunol. 132:2736, 1984), which can also prevent induction of systemic immunological tolerance to an antigen when orally administered simultaneously with the unrelated antigen (Elson, C. O. et al., J. Immunol. 133:2892, 1984).
Based on these observations, mucosal administration of antigens coupled to mucosa-binding molecules such as cholera toxin (or its mucosa-binding fragment, cholera toxin B subunit), has been proposed as a strategy to induce local and systemic immune responses rather than systemic tolerance (McKenzie, S. J. et al., J. Immunol. 133:1818-1824, 1984; Nedrud, J. G. et al., J. Immunol. 139:3484-3492, 1987; Czerkinsky, C. et al., Infect. Immun. 57:1072-1077, 1989; de Aizpurua, H. J. et al., J. Exp. Med. 167:440-451, 1988; Lehner, T. et al., Science 258(5036):1365-1369, 1992).
The present invention provides an immunological tolerance-inducing agent comprising a mucosa-binding molecule linked to a specific antigen derived from an infectious microorganism. The infectious microorganism may be any microorganism, including bacteria, viruses, fungi, helminths, and protozoa, that causes an unwanted immune response, particularly delayed-type hypersensitivity (DTH), in a host following infection. The antigens used for inducing tolerance, called tolerogens, may comprise components of the microorganisms that contain DTH epitopes, i.e., structural determinants that stimulate a DTH response.
The mucosa-binding molecules used to form the tolerance-inducing agent may be derived from viral attachment proteins, lectins, and bacterial fimbriae, although preferred mucosa-binding molecules comprise pure cholera toxin B subunit, pure E. coli heat labile enterotoxin B subunit, or mucosa-binding fragments thereof.
The mucosa-binding molecules and the tolerogens may be linked to each other directly or indirectly, and the linkage may be covalent or non-covalent. For example, the tolerogen may be chemically cross-linked to a mucosa-binding molecule, or may be genetically fused to amino acid sequences comprising the mucosa-binding molecule. Alternatively, the tolerogen may be linked to the mucosa-binding molecule via a spacer molecule, such as a bifinctional antibody.
In another aspect, the present invention encompasses methods for inducing in an individual tolerance to a specific antigen derived from an infectious microorganism. The method is carried out by administering to the individual the tolerance-inducing agent as above, in an amount effective to induce tolerance.
All patents, patent applications, and references referred to in this specification are hereby incorporated by reference in their entirety. In the case of inconsistencies, the present disclosure, including definitions, will prevail.
The present invention is directed towards methods and compositions for inducing immunological tolerance to antigens derived from infectious microorganisms, in particular, antigens that cause an unwanted immune response in a host following infection with the microorganism. The compositions comprise a mucosa-binding molecule linked to a specific microbial antigen. Contrary to established opinion that mucosal administration of antigens coupled to mucosa-binding molecules induces local and systemic immune responses without inducing tolerance, the present inventors have unexpectedly found that mucosal administration of antigens coupled to mucosa-binding molecules according to the present invention induces systemic immunological tolerance to the antigens.
As used herein, xe2x80x9cimmunological tolerancexe2x80x9d refers to a reduction in immunological reactivity of a host towards a specific antigen or antigens. The antigens comprise immune determninants that, in the absence of tolerance, cause an unwanted immune response, such as, for example, acute or chronic inflammation caused by delayed-type hypersensitivity (DTH). DTH is characterized by an immune response at the site of exposure to antigen, which comprises an initial infiltration of neutrophils followed by accumulation of T lymphocytes and blood monocytes, deposition of fibrin, and induration. xe2x80x9cAntigensxe2x80x9d as used herein include haptens, which are compounds that do not stimulate a primary immune response but may trigger a DTH response in a pre-sensitized animal.
An antigen that, when incorporated into a tolerance-inducing agent according to the present invention, promotes the development of tolerance, e,g., the prevention or reversal of DTH, is referred to herein as a xe2x80x9ctolerogenxe2x80x9d. Tolerogens typically comprise xe2x80x9cDTH epitopesxe2x80x9d, which are the particular structural determinants that, in the absence of tolerance, stimulate a DTH response. DTH epitopes may be distinct from xe2x80x9cIgA epitopesxe2x80x9d, which are structural determinants that stimulate the local production of antigen-specific IgA by B lymphocytes in the mucosa. Preferably, tolerogens used in practicing the present invention comprise DTH epitopes and not IgA epitopes.
The present invention encompasses microbial antigens that cause an unwanted immune response in an individual subsequent to infection of the individual by the microbe. The antigens may comprise any component ofthe microorganism that, for example, carries DTH epitopes and excludes IgA epitopes. The antigens may comprise without limitation proteins, peptides, carbohydrates, lipids, nucleic acids, and combinations thereof. Preferably, the antigens comprise peptides or proteins.
The microorganisms from which the antigens are derived may be any microorganism that causes an unwanted immune reaction, e.g., DTH, following infection. The microorganism may be a bacterium, a virus, a fungus, or a parasite such as a helminth or a protozoan. Examples of such microorganisms include without limitation Mycobacterium tuberculosis, Mycobacterium avium, Listeria monocytogenes, Brucella abortus, Chlamydia trachomatis, Mycoplasma sp., Porphyromonas (Bacteroides) gingivalis, Helicobacter pylori, Salmonella sp., Shigella sp., Yersinia sp. Cryptosporidium sp., Borrelia sp., Pneumocystis carinii, Candida albicans, Histoplasma capsulatum, Cryptococcus neoformans, Leishmania sp., Plasmodium, Trypanosoma, paramnyxoviruses (such as respiratory syncytial virus), adenovirus, poliovirus, hepatitis virus, vaccinia and other poxviruses, rhinovirus, herpes simplex virus, variola, and measles virus.
Examples of specific microbial antigens that cause an unwanted immune response in an individual, and which may comprise the specific tolerogen in the immunological tolerance-inducing agent of the present invention, include without limitation: 1) Bacterial toxins or fragments thereof. For example, septicemia-like toxic shock syndrome is induced by systemic exposure to endotoxins (a group of lipopolysaccharides produced by gram negative bacteria) or to certain exotoxins such as staphylococcal enterotoxins; 2) Heat-shock proteins or fragments thereof. As used herein, xe2x80x9cheat shock proteinxe2x80x9d encompasses members of different families of stress proteins (some of which are synthesized constitutively), including without limitation, hsp 60s, hsp 70s, and hsp90s. For example, mycobacterial hsp 55 causes a granulomatous DTH reaction directed against mycobacteria that develops in the lung during tuberculosis. Similarly, chlamydial cells may secrete a 57-60 kDa heat shock protein (hsp), which elicits a DTH reaction in genital and ocular tissues leading to pelvic inflammation and trachoma, respectively; 3) A surface component of a bacteria, virus, fungus, or other microbe, or fragments thereof. For example, the F protein of respiratory syncytial virus comprises a prominent epitope that triggers DTH reactions in infected lung tissue.
It will be understood by those skilled in the art that other antigens suitable for incorporation in the tolerance-inducing agents of the present invention can be purified from their respective microorganisms using well-known methods. For example, whole killed microorganisms can be disrupted and fractionated into component subcellular fractions, using standard chromatographic or electrophoretic methods. The fractions can then be assayed for the presence of DTH epitopes in the following manner: T-cells reactive with the microorganism are obtained from the blood of infected or convalescent patients, or from the spleen or lymph nodes of experimentally infected animals. The T cells are reacted in cell culture with subcellular fractions (or with partially purified or purified components derived therefrom), after which T-cell proliferation is quantified using standard techniques such as, for example, incorporation of 3H-thymidine into DNA. Alternatively, the stimulation of production of cytokines such as, for example, interleukin-2, can be measured after exposure of the T cell population to the cell fractions or components. A DTH epitope derived from the microorganism is identified by the stimulation of 3H-thymidine incorporation or of interleukin-2 production in the T-cell culture.
Another method for identifiing microbial components that contain DTH epitopes is to systemically immunize an experimental animal such as, for example, a mouse, with the microorganism (in an emulsion with an adjuvant such as Freund""s) via a subcutaneous or intraabdominal route. After maintaining the mouse for a sufficient time to induce a systemic immune reaction, the potential DTH epitope-containing fraction or component is then injected into the footpad of the animal, and the appearance of a DTH reaction is monitored.
According to the present invention, antigens comprising a DTH epitope are linked to a mucosa-binding molecule to form an immunological tolerance-inducing agent. Mucosa-binding molecules useful in practicing the present invention include without limitation mucosa-binding subunits or domains of bacterial toxins, bacterial fimbriae, viral attachment proteins, and lectins. Non-limiting examples of these types of mucosa-binding molecules are listed in Table 1.
Preferred mucosa-binding molecules are mucosa-binding subunits or domains of bacterial toxins such as cholera toxin or E. coli heat-labile enterotoxin; most preferred is pure cholera toxin B subunit (CTB). xe2x80x9cPurexe2x80x9d as used in this context means that the cholera toxin B subunit is essentially free of detectable contamination by active cholera toxin, which comprises a cholera toxin A subunit (CTA) in combination with CTB. An xe2x80x9cactivexe2x80x9d cholera toxin molecule as used herein denotes one that exhibits ADP-ribosylating activity. Functional purity of cholera toxin B subunit for use in the present invention can be achieved by expressing the gene encoding cholera toxin B subunit in a bacterial cell (such as, for example, E. coli or V. cholerae) in the absence of a gene encoding the cholera toxin A subunit. Methods for large-scale expression of pure cholera toxin B subunit are disclosed in, for example, U.S. Pat. No. 5,268,276. As shown in Example 6 below, the present inventors have found that the presence of even small amounts of contaminating cholera toxin A subunit can abrogate the tolerance induced by the compositions of the present invention. The present invention also encompasses mucosa-binding fragments of, e.g., CTB or LTB.
In practicing the present invention, the tolerogen and the mucosa-binding molecule may be linked to each other directly or indirectly. In both cases, the linkages may be covalent or non-covalent (e.g., via electrostatic and/or hydrophobic interactions).
In one embodiment, direct linkage between the tolerogen and mucosa-binding molecule is achieved by chemically cross-linking the tolerogen and the mucosa-binding moiety. It will be understood that any suitable method may be used to cross-link the components, as long as the final cross-linked product retains the ability to induce tolerance to the specific antigen employed. Suitable chemical cross-linking procedures are well-known in the art; see, for example, Carlsson J. et al., Biochem. J. 173:723-737, 1978; Cumber, J. A. et al. Methods in Enzymology 112:207-224, 1985; Walden, P. et al., J. Mol. Cell Immunol. 2:191-197, 1986; Gordon, R. D. et al., Proc. Natl. Acad. Sci. (USA) 84:308-312,1987; Avrameas, S. etal., Immuno-chemistry 6:53,1969; Joseph, K. C. et al., Proc. Natl. Acad. Sci. USA 75:2815-2819, 1978; Middlebrook, J. L. et al., Academic Press, New York, pp. 311-350, 1981).
In another embodiment, direct linkage is achieved by the design and expression of a recombinant chimeric gene encoding a fusion protein that comprises the tolerogen, or a fragment thereof containing a DTH epitope, which is fused to a mucosa-binding peptide or polypeptide (Sanchez et al., FEBS Letts. 241:110, 1988). The chimeric gene is then expressed in a suitable expression system, including without limitation bacteria, yeast, insect cells, or mammalian cells, and the hybrid protein gene product isolated therefrom.
Indirect linkage between the tolerogen and the mucosa-binding molecule may be achieved using a spacer molecule. Preferably, the spacer molecule has an affinity for either the tolerogen, the mucosa-binding molecule, or both. In one embodiment, the spacer comprises an antibody, preferably a bifunctional antibody that recognizes both the tolerogen and the mucosa-binding molecule. In another embodiment, the spacer molecule is derived from the cholera toxin-binding structure of the GM1 ganglioside, galactosyl-N-acetyl-galactosaminyl-(sialyl)-galactosylglucosylceramide. In these cases, the linkages are formed by high-affinity binding between the spacer and the other components. The only requirement is that the tolerogen and mucosa-binding molecule both remain linked to each other via the spacer during mucosal administration. In the case of spacer molecules that do not have specific affinity for either tolerogen or mucosa-binding molecule, chemical cross-linking methods may be used as described above to form covalent linkages between the components. In another embodiment, an indirect linkage may be achieved by encapsulating the tolerogen within a protective vehicle such as a liposome (or equivalent biodegradable vesicle) or a microcapsule, on the surface of which the mucosa-binding molecule is arrayed. In this type of presentation form, the tolerogen may be free within the lumenal space of the vesicle or microcapsule, or may be bound to other components. The only requirement is that the tolerogen and the mucosa-binding molecule remain in close enough proximity during mucosal administration such that the tolerogen is effectively delivered to the mucosa.
In yet another embodiment, the tolerance inducing agent comprises a nucleic acid sequence encoding the tolerogen, which is then chemically coupled to the mucosa-binding molecule and administered by the mucosal route. xe2x80x9cNucleic acidxe2x80x9d as used herein denotes DNA, both single- and double-stranded, with a sugar backbone of deoxyribose, methylphosphonate, or phosphorothioate; xe2x80x9cprotein nucleic acid: (PNA), which comprises nucleotides bound to an amino acid backbone; and all forms of RNA. This method requires cells in the host mucosal tissues to transcribe and translate the corresponding gene into a mature peptide or protein (Rohrbaugh, M. L. et al, N. Y. Acad. Sci. 685:697-712, 1993; Nabel, G. J. et al., Trends in Biotechnology Vol. 11, No. 5, pp. 211-215, 1993; Robinson, H. L. et al., Vaccine 11:957-960, 1993; Martinon, F. et al., Eur. J. Immunol. 23:1719-1722, 1993).
The present invention is also directed to a method of inducing in an individual immunological tolerance against a specific microbial antigen that causes an unwanted immune response, such as, for example, DTH. The method comprises administering to the individual by a mucosal route an immunological tolerance-inducing agent as described above.
The methods ofthe present invention can be practiced preventively or therapeutically. That is, the timing of administration relative to the time of exposure to microbial antigens is not limiting. For example, the tolerance inducing agent can be administered to individuals who have been deemed xe2x80x9cat-riskxe2x80x9d for developing immune-mediated tissue damage caused by tuberculosis, or to patients who have already exhibited clinical indication of tuberculosis. Similarly, women harboring chlamydia infections are often asymptomatic and thus the agent could be given preventively or after the infection has been diagnosed.
Examples of pathological syndromes to which the methods and compositions of the present invention may be applied include, without limitation, tuberculosis, chlamydial infections, schistosomiasis, leprosy, pneumocystis pneumonia, leishmaniasis, and infections by Candida albicans, Plasmodium, Trypanosoma, Listeria monocytogenes, Brucella abortus, mycoplasma sp., Porphyromonas (Bacteroides) gingivalis, Helicobacter pylori, Salmonella sp., Shigella sp., Yersinia sp., Histoplasma capsulatum, Cryptococcus neoformans, Cryptosporidium sp., Borellia sp., as well as by the following viruses: paramyxoviruses such as respiratory syncytial virus, adenovirus, poliovirus, hepatitis virus, vaccinia and other poxviruses, rhinovirus, herpes simplex virus, variola, and measles virus.
It is also contemplated that the present methods can be used to induce tolerance against live microorganisms (recombinant or native) used for delivery of vaccinal antigens. Examples include recombinant live bacteria, e.g., BCG, Salmonella, Shigella, Lactobacillus; and viruses, e.g., adenovirus, poliovirus, poxviruses, Semliki Forest Virus, and retroviruses.
Target tissues suitable for mucosal administration according to the present invention include without limitation the gastrointestinal tract (including the mouth and throat), the respiratory tract (including the nose), the eye, and the genital tract. Thus, tolerance-inducing agents or compositions are formulated into dosage unit forms for mucosal administration, such as for example, creams, ointments, lotions, powders, liquids, tablets, capsules, suppositories, sprays, or the like. Dosage unit forms can include, in addition, one or more pharmaceutically acceptable excipient(s), diluent(s), disintegrant(s), lubricant(s), plasticizer(s), colorant(s), dosage vehicle(s), absorption enhancer(s), stabilizer(s), bactericide(s), or the like. One or more immunologically active substances that enhance the tolerogenic activity of these formulations may also be included, e.g., cytokines such as interleukin-10 (IL-10), interleukin-4 (IL-4), and transforming growth factor-beta (TGF-xc3xa2).
The tolerance-inducing agents are present in the dosage forms such that a single dosage unit contains between about 1 ig and about 10 mg of the agent, preferably between about 10 ig and about 1 mg. Each dosage unit may contain an amount of active agent effective to induce tolerance. Alternatively, the dosage unit form may include less than such an amount, if multiple dosage unit forms or multiple dosages are to be used to administer a total dosage of the active agent.
It will be understood that the administration regimen for prevention or treatment of an unwanted immune response caused by an infectious microorganism will depend upon the particular organism (and immune response), as well as on the dosage form used. Without wishing to be bound by theory, it is contemplated that effective dosages will be much lower than those employed with tolerogen alone. An administration regimen effective in preventing or treating a particular unwanted immune response can be determined by experimentation known in the art, such as by establishing a matrix of dosages and frequencies and comparing a group of experimental units or subjects to each point in the matrix. Specifically, blood is obtained from experimental subjects and lymphocytes are isolated therefrom. The lymphocytes are then exposed to antigen alone (i.e., in the absence of a mucosa-binding molecule), and T-lymphocyte proliferation is measured as described above (e.g., by incorporation of 3H-thymidine). The efficacy of the tolerization method is indicated by a lessening of lymphocyte proliferation in response to antigen relative to controls. Similarly, the efficacy of tolerization may be monitored by measuring the production of cytokines such as interleukin-2 (IL-2) by the lymphocytes in response to antigen. In this case, the less IL-2 produced, the more effective the induction of tolerance.
The present invention is exemplified by the use of cholera toxin B subunit (CTB) and E. coli heat-labile enterotoxin B subunit (LTB) as mucosa-binding molecules, and the use of sheep red blood cells (SRBC) and human gamma-globulins (HGG) as antigens/tolerogens. Though neither antigen is derived from a microorganism, these antigens are excellent models of particulate and soluble antigens, respectively. They are among the best characterized oral tolerogens with regard to both antibody formation and cell-mediated immune reactions, the latter reactions being typified by the classical delayed-type hypersensitivity (DTH) reaction.
The following experiments are provided for the purpose of illustrating the subject invention but in no way limit its scope.
Materials and Methods
Inbred Balb/c female mice were obtained from the Animal Care Facility of the Department of Medical Microbiology and Immunology, University of Gxc3x6teborg, Sweden. Mice 6-8 weeks of age were used.
Purification of the Mucosa-binding Molecules CTB and LTB
Recombinant cholera toxin B subunit (CTB) was produced in a mutant strain of Vibrio cholerae deleted of the cholera toxin genes and transfected with a plasmid encoding the CTB subunit (Sanchez, J. et al., Proc. Natl. Acad. Sci USA 86:481-485, 1989). Recombinant B subunit of Escherichia coli heat-labile enterotoxin (LTB) was produced in a similar mutant strain of Vibrio cholerae deleted of the cholera toxin genes and transfected, in this case, with a plasmid encoding E. coli LTB (Hirst T. R., et al., Proc. Natl. Acad. Sci. USA, 81:7752-7756, 1984). In these expression systems, CTB and LTB are recovered from bacterial growth media as secreted proteins. Bacterial cultures were centrifuged at 8000 rpm for 20 min, and the supernatants were collected and adjusted to pH 4.5 with dilute HCl. After precipitation with hexametaphosphate (final concentration 2.5 g/l) for 2 hours at 23xc2x0 C. followed by centrifugation at 8000 rpm, the pellets were resuspended in 0.1 M sodium phosphate buffer, pH 8.0, and were dialysed against 0.01 M phosphate-buffered saline, pH 7.2. The dialysate was then centrifuged at 15 000 rpm to remove residual insoluble material and the supernatant was further clarified by filtration through a 0.22 Fm filter (Millipore, Bedford, Mass.). Finally, CTB and LTB were purified by standard gel filtration chromatography through columns of Sephadex G-100 (Pharmacia, Sweden).
Purification of Human Gamma-globulins (HGG)
HGG was purified from pooled human sera by sequential precipitation with (NH4)2SO4 (final concentration 40%), followed by gel filtration chromatography on a column of Sephacryl S-300 HR (Pharmacia, Sweden) previously equilibrated with phosphate-buffered saline (0.2 M sodium phosphate, NaCl 0.1 M, pH 8.5). The resulting HGG preparation was diluted to 15 mg/ml.
Preparation of CTB-conjugated Sheep Red Blood Cells (SRBC-CTB)
Sheep red blood cells (SRBC) were stored at 4xc2x0 C. in Alsevier""s solution until use. Immediately prior to use, SRBC were washed 3 times with phosphate-buffered saline (PBS) (0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4) by centrifugation at 3000 rpm for 10 min, and were then resuspended in PBS at a cell density of 5xc3x97109 SRBC/ml. To facilitate coupling of CTB to SRBC, SRBC were first coupled to GM1 ganglioside. A solution of PBS containing 300 nmol/ml GM1 ganglioside (Sigma Chemical Co., St Louis, Mo.) was added to packed SRBC at a ratio of 1:2 (vol/vol), and incubation was carried out at 37xc2x0 C. for 2 hours in a shaking water bath. After 3 washes with PBS to remove excess GM1, GM1-coated red cells were resuspended in PBS to a density of 5xc3x97109 SRBC/ml and mixed with recombinant CTB (Sanchez, J., et al., Proc. Natl. Acad. Sci. USA 86:481-485, 1989) (final concentration 50 Fg/ml). After incubation for 2 hours at 37xc2x0 C. in a shaking water bath to allow binding of CTB to GM1-coated SRBC, the red cell suspension was washed twice with PBS to remove non cell bound CTB, and resuspended in PBS. The final pellet was at a cell density of 1xc3x971010/ml.
To ascertain that the CTB molecules had bound to GM1-coupled SRBC and were still able to bind additional GM1 molecules, a solid phase hemadsorption assay using GM1 immobilized on plastic wells was employed. An aliquot of red cell suspension was diluted in PBS to a final concentration of 1% (packed vol/vol) supplemented with 0.1% (weight/vol) of bovine serum albumin (BSA) (Sigma) and added to GM1-coated U-shaped wells of plastic microtiter plates (Costar). After incubation at ambient (22xc2x0 C.) temperature, wells were examined for appearence of hemadsorption. The specificity of the assay was established by the absence of hemadsorption in control wells that had not been coated with GM1, and by the dose-dependent inhibition of hemadsorptionby the addition of cell-free CTB to GM1-coated wells during incubation with the red blood cells.
Preparation of LTB-conjufated Sheep Red Blood Cells (SRBC-LTB)
GM1-coated SRBC (5xc3x97109 GM1-SRBC/ml) were conjugated to recombinant LTB (50 Fg/ml) exactly as described above for coupling of SRBC to CTB.
Preparation of CTB-conjugated Human Gamma-globulins (HGG-CTB)
CTB and HGG were each coupled to N-succinimidyl (3-(2-pyridyl-dithio) propionate (SPDP) (Pharmacia, Uppsala, Sweden) (Carlsson, J., et al., Biochem. J. 173:723, 1978) at molar ratios of 1:5 and 1:10 respectively. SPDP was added to HGG and the mixture was allowed to incubate for 30 min at 23xc2x0 C. with stirring. Excess SPDP was removed by gel filtration on a column of Sephadex G-25 (Pharmacia, Sweden) equilibrated with acetate buffer (0.1M sodium acetate, 0.1M NaCl, pH 4.5). The SPDP-derivatized HGG was reduced with dithiothreitol (DTT) (final concentration 50 mM) for 20 min at 23xc2x0 C., and the resulting preparation was passed through a column of Sephadex G-25 equilibrated with phosphate-buffered saline (0.2M sodiumphosphate, NaCl 0.1 M, pH 8.5) to remove excess DTT and pyridine-2-thione released during reduction of SPDP-derivatized HGG.
CTB was diluted to 2 mg/ml in PBS and derivatized with SPDP as described above for HGG but at a molar ratio of 5:1 (SPDP:CTB). The resulting SPDP-derivatized CTB was passed through a column of Sephadex G-25 equilibrated in the same buffer, to remove excess unreacted SPDP.
SPDP-derivatized HGG and CTB were mixed at an equimolar ratio and incubated for 16 h at 23xc2x0 C. The resulting CTB-HGG conjugate was purified by gel filtration through a column of Sephacryl S-300 to remove free CTB and/or HGG. The resulting conjugate was shown to contain GM1 ganglioside binding capacity and to retain both CTB and HGG serological reactivities by means of an ELISA using GM1 (Sigma, St Louis, Mo.) as solid phase capture system (Svennerholm, A.-M. et al. Curr. Microbiol. 1:19-23, 1978), and monoclonal and polyclonal antibodies to CTB and HGG as detection reagents (see below). Serial two-fold dilutions of the conjugate and of purified CTB- and HGG-SPDP derivatives were incubated in polystyrene wells that had previously been coated with GM1 ganglioside, and in wells coated with rabbit polyclonal IgG antibodies to HGG; next, horseradish peroxidase (HRP) conjugated rabbit ant-HGG or mouse monoclonal anti-CTB antibodies (appropriately diluted in PBS containing 0.05% Tween 20), followed by enzyme substrate, were applied sequentially to detect solid phase bound HGG and CTB. The amount of free and bound HGG and CTB was determined by reference to standard curves calibrated with known amounts of SPDP derivatized antigens. On average, the SPDP conjugation procedure and purification protocol described above yielded preparations containing negligible amounts of free HGG and less than 10% free CTB.
Immunization Protocols
Immunization with SRBC: Primary systemic immunization: Mice were injected in the rear left footpad with 40 Fl of pyrogen-free saline containing 107 SRBC. Secondary systemic immunization: Five days after the primary immunization, mice were challenged by injecting the right rear footpad with 40 Fl of pyrogen-free saline containing 108 SRBC.
Immunization with HGG: Prior to immunization, HGG was aggregated by heating at 63xc2x0 C. for 30 min. Primary systemic immunization: Mice received 0.2 ml of aggregated HGG (500 Fg) emulsified in Freund""s complete adjuvant (Difco, St Louis, Mo.) and administered by subcutaneous injections into the flanks. Secondary systemic immunization: Five days after the primary immunization, mice were challenged by injecting the right rear footpad with 40 Fl of pyrogen-free saline containing 1 mg of HGG.
Oral tolerance induction protocols: At various times before or after the primary systemic immunization with SRBC, mice were administered a single dose or daily consecutive doses of SRBC or SRBC-CTB. Each dose consisted of 2.5xc3x97109 SRBC or SRBC-CTB in 0.5 ml of PBS given by the intragastric route using a baby catheter feeding tube. Control animals were given 0.5 ml of PBS alone.
For induction of tolerance to HGG, mice were given a single oral dose of unconjugated HGG or CTB-conjugated HGG administered by intragastric tubing, 1 week before primary systemic immunization with HGG. Doses of 1 mg and 5 mg of unconjugated HGG and of 60 Fg of CTB-conjugated HGG were tested.
Evaluation of Delayed-type Hypersensitivity (DTH) Reactions
DTH to SRBC: Thickness of the right footpad was measured immediately before, and 2, 4, 24, and 48 h after the secondary systemic immunization with SRBC, using a dial gauge caliper (Oditest, H. C. Kxc3x6plin, Schluchtem, Essen, Germany). The intensity of DTH reactions was determined for each individual animal by substracting the value obtained before challenge from those obtained at various times after challenge.
DTH to HGG: The intensity of DTH reactions to HGG injected in the right footpad was evaluated as above for SRBC.
Evaluation of Serum Antibody Responses
Serum anti-SRBC antibody responses: Immediately before the primary systemic immunization with SRBC administered in the left footpad, and 1-2 weeks after the secondary systemic immunization, a sample of blood was collected from the tail vein of individual mice and allowed to clot at room temperature for 60 min. Sera were heated at 56xc2x0 C. for 45 min to inactivate complement, and then assayed for antibody levels to SRBC by direct and indirect hemagglutination assays. For direct hemagglutination, serial 2-fold dilutions of serum samples in PBS supplemented with 0.1% (weight/vol) of bovine serum albumin (PBS-BSA) were prepared in U-bottom wells of microtiter-plates. Fifty microliters of a suspension of 0.5% (packed vol/vol) SRBC in PBS-BSA were added to all wells and the plates were incubated for 1 hour at ambient temperature followed by an overnight incubation at 4xc2x0 C. Wells were then examined for hemagglutination.
To detect non-hemagglutinating antibodies that had bound to SRBC, 25 Fl of PBS containing a mixture of heat-inactivated (56xc2x0 C. for 45 min) rabbit antisera to mouse IgG and mouse IgA (final dilution 1:50) were added to wells corresponding to serum dilutions shown to be negative in the direct hemagglutination assay. The plates were then shaken to allow resuspension of SRBC and incubated undisturbed at 4xc2x0 C. for 2 hours. Thereafter the wells were examined for hemagglutination. The reciprocal of the highest dilution of any given mouse serum causing hemagglutination of SRBC either directly or after addition of anti-mouse antisera (in the indirect hemagglutination assay) was determined and defined as the anti-SRBC antibody titer of said mouse serum.
Serum anti-HGG antibody responses: Serum IgM and IgG antibody levels to HGG were determined by standard solid phase ELISA using polystyrene microwells coated with HGG as solid phase capture system and HRP-conjugated affinity purified goat antibodies to mouse IgG and to mouse IgM (Southern Biotechnology Associates, Birmingham, Ala.) as detection reagents. Serial 5-fold dilutions of mouse sera were prepared in PBS containing 0.05% Tween 20 and incubated for 2 hrs at 23xc2x0 C. in HGG-coated wells. After 5 washings with PBS containing 0.05% Tween 20, appropriately diluted HRP-antibodies to mouse IgM or IgG were added. Two hours later, plates were rinsed with PBS, and solid phase bound enzyme activity was revealed by addition of chromogen substrate, consisting of ABTS tablets (Southern Biotechnology Associates) dissolved in citrate-phosphate buffer, pH 5.0 and containing H2O2. Absorbance values were monitored 30 min later with an automated spectrophotometer (Titerscan, Flow Laboratories). The anti-HGG antibody titer of a mouse serum was defined as the reciprocal of the highest dilution given an absorbance value of at least twice that of control wells exposed to buffer alone instead of serum.
In vitro lymphocyte proliferation assay: Lymph nodes obtained 1-2 weeks after the secondary systemic immunization were minced in Iscove""s medium (Gibco Europe, U.K.) and pressed through sterile nylon-mesh screens to yield single cell suspensions. The cells were washed twice and resuspended at 2xc3x97106 cells/ml in Iscove""s medium supplemented with 5% heat-inactivated fetal bovine serum (FBS), L-glutamine (1%), sodium pyruvate (1%), non-essential aminoacids (1%), 2-mercaptoethanol (5xc3x9710xe2x88x925 M) and gentamycin (20 Fg/ml). Lymph node cells were added to flat-bottom microtiterwells (Nunc, Denmark) containing a previously titrated amount of SRBC in a total volume of 200 Fl. The plates were then incubated at 37xc2x0 C. in 5% CO2 in air for 3 days. The cultures were pulsed during the last 16 hrs with 3H-thymidine (2.0 mCi/mM, Amersham, Stockholm), individual wells were harvested using a 96-well automated cell-harvester (Inotech, Basel, Switzerland) and the radio-nucleotide incorporation was measured with an argon-activated scintil-lation counter (Inotech).
The level of 3H-thymidine incorporation was calculated as the stimulation index (S.I.)=CPM of lymph node cells+SRBC/CPM of lymph node cells alone.